Novel Immunointeractive Molecules and uses Thereof

ABSTRACT

The present invention relates generally to molecules such as peptides, polypeptides and proteins which interact immunologically with T lymphocytes in subjects having peanut allergy, or allergy to other tree nuts, and genetic sequences encoding same. These molecules are preferentially immunointeractive with T cells in subjects having an allergy to the Ara h 2 allergen. The molecules of the present invention are useful in the development of diagnostic, therapeutic and prophylactic agents for conditions characterised by an aberrant, inappropriate or otherwise unwanted immune response to Ara h 2 or derivative or homologue thereof.

FIELD OF THE INVENTION

The present invention relates generally to molecules such as peptides,polypeptides and proteins which interact immunologically with Tlymphocytes in subjects having peanut allergy, or allergy to other treenuts, and genetic sequences encoding same. These molecules arepreferentially immunointeractive with T cells in subjects having anallergy to the Ara h 2 allergen. The molecules of the present inventionare useful in the development of diagnostic, therapeutic andprophylactic agents for conditions characterised by an aberrant,inappropriate or otherwise unwanted immune response to Ara h 2 orderivative or homologue thereof.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected alphabetically at the end of thedescription.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

Peanut allergy is a life-threatening and incurable disorder, affectingapproximately 1% of the general population (Sicher et al., J AllergyClin Immunol 103: 559-562, 1999). It is characterised by the suddenonset of anaphylaxis, which may occur with exposure to minute quantitiesof peanut proteins (Hourihane et al., J Allergy Clin Immunol 100:596-600, 1997). Nut induced anaphylaxis is that most frequentlyassociated with mortality or with life-threatening features (Sampson etal,. N Engl J Med 327: 380-4, 1992). Peanut proteins are frequentlyconcealed within apparently safe food sources, such that accidentalcontact occurs for up to 50% of sufferers over a 5 year period (Sichereret al., Paediatrics 102: e6, 1998). Not surprisingly, nut allergy isassociated with significant psychological morbidity for sufferers andcarers alike, akin to that suffered by those with chronic debilitatingillnesses such as rheumatoid arthritis (Primeau et al., Clin Exp Allergy30: 1135-43,2000). Cure, while being an imperative to remove nut allergyas a cause of mortality, is also necessary to remove the chronicpsychological burden that peanut allergic subjects carry.

To date, efforts at immunotherapy for peanut allergy have been met byextremely limited success. Nelson et al. have shown that tolerance ofpeanut can be induced using a rush immunotherapy protocol, but thattolerance is lost in approximately half of the subjects duringmaintenance dosing and additionally that injections are associated withfrequent episodes of anaphylaxis in the majority of subjects during boththe buildup and maintenance phases (Nelson et al., J Allergy ClinImmunol 99: 744-51, 1997). Oppenheimer et al. demonstrated similarfindings within their study, again showing that active therapy isassociated with a high rate of systemic anaphylaxis. Data collection inthat study was terminated after the administration of peanut extract toa placebo. randomised subject resulted in their death, highlighting thedangerous nature of this condition (Oppenheimer et al., J Allergy ClinImmunol 90: 256-62, 1992).

Development of novel strategies to overcome the morbidity associatedwith allergen immunotherapy depends on an accurate understanding of theimmunological basis to successful immunotherapy, as well as itsside-effects. It has long been established that morbidity due toallergen immunotherapy is due to the cross-linking of IgE, and that thisaction is not required for such therapy to be efficacious (Litwin etal., Int Arch Allergy Appl Immunol 87: 361-61,998). It is also knownthat one of the critical actions of immunotherapy in producing toleranceis its ability to change the predominant specific T cell phenotype froma T_(H)2 to a T_(H)1 phenotype (Robinson, Br Med Bull 56: 956-968,2000).Although the precise pathway through which this change occurs remainsundocumented, current theories suggest that this is likely to occur viathe suppression of the T_(H)2 phenotype by IL-10, then reconstitution ofa normal immune response via the actions of IL-2 and IL-15 (Akdis etal., Allergy 55: 522-530, 2000).

A key difference in antibody and lymphocyte responses is in antigenrecognition, antibodies recognising conformational epitopes dependent onmolecular tertiary structure, while CD4+ T cells recognise short linearpeptides. This difference in antigen recognition is the basis to manynovel strategies of immunotherapy, including that using peptides basedupon T cell epitopes, B cell epitope mutants and altered peptide ligands(Akdis et al., Trends Immunol 22: 175-8, 2001). Such methods all dependon the alteration or absence molecular tertiary structure, so that IgEcross-linking and effector cell activation is lost. Peptideimmunotherapy is the method for which the best evidence of efficacyexists, being documented for both cat dander allergy and bee venomallergy. Muller et al. (1998) showed that, in the absence of anysystemic side-effects, tolerance could be achieved for the major beevenom allergen Phospholipase A2 (PLA2) using sequences based on itsthree major epitopes, while several authors have demonstrated thatpeptides based on the structure of the major cat allergen Fel d 1 can beused to induce diminished-clinical responses (Muller et al., J AllergyClin Immunol 101: 747-754, 1998; Norman et al., Am J Respir Crit CareMed 154: 1623-8, 1996; Marcotte et al., J Allergy Clin Immunol 101:506-13, 1998; Pene et al., J Allergy Clin Immunol 102: 571-8, 1998;Maguire et al., Clin Immunol 93: 222-31, 1999). Crucial to thedevelopment of such strategies is the retention of T cell epitopes, sothat T cell phenotypic change can be induced.

Accordingly, there is a need to both identify the major peanut allergensand, further, to identify the T cell epitopes of these allergens. Theidentification characterisation, and analysis of these epitopes iscritical to the development of specific diagnostic and immunotherapeuticmethodology.

In work leading up to the present invention, the inventors haveidentified the human T cell epitopes of the peanut allergen, Ara h 2.The identification of Ara h 2 T cell epitopes now facilitates thedevelopment of molecules and methodology for the diagnosis and treatmentof conditions characterised by the aberrant, inappropriate or otherwiseunwanted immune response to Ara h 2 or derivative or homologue thereofsuch as peanut allergy or other tree-nut allergy.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The subject specification contains amino acid and nucleotide sequenceinformation prepared using the programme Patent In Version 3.1,presented herein after the bibliography. Each amino acid or nucleotidesequence is identified in the sequence listing by the numeric indicator<201 > followed by the sequence identifier (eg. <210>1, <210>2, etc).The length, type of sequence (DNA, protein, etc) and source organism foreach amino acid or nucleotide sequence is indicated by informationprovided in the numeric indicator fields <211>, <212> and <213>,respectively. Amino acid and nucleotide sequences referred to in thespecification are identified by the indicator SEQ ID NO: followed by thesequence identifier (eg. SEQ ID NO:1, SEQ ID NO:2, etc.). The sequenceidentifier referred to in the specification correlates to theinformation provided in numeric indicator field <400> in the sequencelisting, which is followed by the sequence identifier (eg. <400>1,<400>2, etc). That is SEQ ID NO:1 as detailed in the specificationcorrelates to the sequence indicated as <400>1 in the sequence listing.

One aspect of the present invention provides an isolated peptide of theformula:X₁X₂X₃

wherein:

-   -   X₁ and X₃ may be the same or different and each is an amino acid        sequence comprising from 0 to 40 naturally or non-naturally        occurring amino acid residues;    -   X₂ is any amino acid sequence derived from or homologous to Ara        h 2;

and wherein said peptide molecule is capable of interacting with T cellsand modifying T cell function when incubated with cells from subjectshaving a condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,analogue, mutant, chemical equivalent or mimetic of said peptide.

Another aspect of the present invention provides an isolated peptide ofthe formula:X₁X₂X₃

wherein:

-   -   X₁ and X₃ may be the same or different and each is an amino acid        sequence comprising from 0 to 40 naturally or non-naturally        occurring amino acid residues;    -   X₂ is an amino acid sequence of from 5 to 100 residues derived        from, homologous to or contiguous with amino acids 1-157        inclusive or derivatives thereof of Ara h 2;

and wherein said peptide molecule is capable of interacting with T cellsand modifying T cell function when incubated with cells from subjectshaving a condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,mutant, chemical equivalent or mimetic of said peptide.

Yet another aspect of the present invention provides an isolated peptideof the formula:X₁X₂X₃

wherein

-   -   X₁ and X₃ may be the same or different and each is an amino acid        sequence comprising from 0 to 40 naturally or non-naturally        occurring amino acid residues;    -   X₂ is an amino acid sequence of from 5 to 100 residues derived        from, homologous to or contiguous with amino acids 19-92, 91-119        and/or 127-155 inclusive or derivatives thereof of Ara h 2;

and wherein said peptide molecule is capable of interacting with T cellsand modifying T cell function when incubated with cells from subjectshaving a condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,analogue, mutant, chemical equivalent or mimetic of said peptide.

Still another aspect of the present invention provides a peptide, ashereinbefore defined, wherein the antibody reactivity of said peptide isinhibited, abrogated or otherwise down-regulated.

Yet another aspect of the present invention provides an isolated peptidecomprising any amino acid sequence derived from or homologues to Ara h 2wherein said peptide molecule is capable of interacting with T cells andmodifying T cell function when incubated with cells from subjects havinga condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,analogue, mutant, chemical equivalent or mimetic of said peptide.

A further aspect of the present invention provides an isolated peptidecomprising an amino acid sequence of from 5-100 residues derived from,homologues to or contiguous with amino acids 1-157 inclusive orderivatives thereof of Ara h 2 wherein said peptide molecule is capableof interacting with T cells and modifying T cell function when incubatedwith cells from subjects having a condition characterised by anaberrant, unwanted or otherwise inappropriate immune response to Ara h 2or a derivative, homologue, analogue, mutant, chemical equivalent ormimetic of said peptide.

In yet another aspect, the present invention provides a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding the peptides as hereinbefore defined or aderivative, homologue or analogue thereof.

In still another aspect the present invention provides a method for thetreatment and/or prophylaxis of a condition in a subject, whichcondition is characterised by the aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2, said method comprisingadministering to said subject an effective amount of a peptide ashereinbefore defined for a time and under conditions sufficient toremove or reduce the presence or function in said subject of T cellsdirected to said Ara h 2.

A further aspect of the present invention contemplates the use of anagent as hereinbefore defined in the manufacture of a medicament for thetreatment of a condition in a mammal, which condition is characterisedby an aberrant, unwanted or otherwise inappropriate immune response toAra h 2.

In yet another further aspect, the present invention contemplates apharmaceutical composition comprising an agent as hereinbefore definedand one or more pharmaceutically acceptable carriers and/or diluents.Said agents are referred to as the active ingredients.

Yet another aspect of the present invention relates to agents, ashereinbefore defined, when used in the method of the present invention.

Still another aspect of the present invention is directed to a method ofdiagnosing or monitoring a condition in a mammal, which condition ischaracterised by an aberrant, unwanted or inappropriate response to Arah 2, said method comprising screening for Ara h 2 reactive T cellsutilising the peptides hereinbefore defined.

In another aspect the present invention provides diagnostic kits for usein the diagnostic methodology hereinbefore defined.

Single and three letter abbreviations used throughout the specificationare defined in Table 1. TABLE 1 Single and three letter amino acidabbreviations Three-letter One-letter Amino Acid Abbreviation SymbolAlanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp DCysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of Ara h 2 20-mer peptide series.Peptides of 20 amino acids in length, overlapping by 11 amino acidsexcept for those at the N-terminus (18 amino acid overlap), representingthe entire amino acid sequence of Ara h 2 were used. The residue numbersof each peptide is shown in the left hand column, while overlap withpeptides located adjacent to each peptide is demonstrated by the extentto which each peptide is overlaid within the figure. Peptide sequencesare based upon the Ara h 2 sequence described by Stanley et al (Stanleyet al, Arch Biochem Biophys. 342: 244-253, 1997) (SEQ ID NO: 1).

FIG. 2 is a graphical representation of the evaluation of T cellmitogenic and cytotoxic potential of Ara h 2 dodecapeptides.

a. Mitogenicity Assay

Triplicate cultures of 5×10⁴ cells/well of a 3 week oligoclonalHDM-specific TCL together with 5×10⁴ cells/well of irradiated autologousPBMC and Ara h 2 peptides at a concentration of 10 μg/ml for 72 hours.For the last 12 hours cells were pulsed with ³H-thymidine beforeharvesting and counting. ³H-thymidine incorporation for each peptide wasestimated and expressed as mean cpm+SEM.

b. Toxicity Assay

Triplicate cultures of 5×10⁴ cells/well of a 3 week oligoclonalHDM-specific TCL stimulated with 50 IU/ml of recombinant human IL-2 andAra h 2 peptides at a concentration of 10 μg/ml for 72 hours. For thelast 12 hours cells were pulsed with 0.1 mCu/well ³H-thymidine beforeharvesting and counting. ³H-thymidine incorporation for each peptide wasestimated and expressed as mean cpm+SEM.

FIG. 3 is an image of a Western blot for serum IgE reactivity to Ara h 2of peanut allergic and non-allergic subjects.

Legend: M, molecular mass; NS, no serum.

Following resolution of CPE on a 14% polyacrylamide gel, proteins weretransferred to nitrocellulose and probed with sera from the studypopulation, and non-peanut allergic controls. IgE was detected using HRPconjugated mouse anti-human IgE and enhanced chemiluminescence.

FIG. 4 is a graphical representation of peanut allergic donor peanutspecific TCL proliferative responses to Ara h 2 peptides, Ara h 2 andcrude peanut extract. Peanut specific TCL (5×10⁴ cells/ml) fromindividuals with peanut allergy generated using CPE were stimulated withAra h 2 peptides at a concentration of 10 μg/ml in the presence ofautologous irradiated PBMC as APC (5×10⁴ cells/ml) in 3 day cultures.Proliferative responses were assessed by tritiated thymidineincorporation, and displayed as mean cpm+SEM of triplicate cultures.

FIG. 5 is a graphical representation of peanut allergic donor peanutspecific Ara h 2 pulsed TCL proliferative responses Ara h 2 peptides,Ara h 2 and crude peanut extract. Peanut specific TCL (5 x 10⁴ cells/ml)from individuals with peanut allergy were generated using CPE and rAra h2, then stimulated with Ara h 2 peptides at a concentration of 10 pg/mlin the presence of autologous irradiated PBMC as APC (5×10⁴ cells/ml) in3 day cultures. Proliferative responses were assessed by tritiatedthymidine incorporation, and displayed as mean cpm+SEM of triplicatecultures.

FIG. 6 is a graphical representation of the percentage responderfrequency to Ara h 2 peptides amongst peptide responsive CPE driven TCL.The percentage of peptide responsive TCLs demonstrating a stimulationindex of ≧2.5 to individual peptides is demonstrated, along withresponses to rAra h 2 and CPE.

FIG. 7 is a graphical representation of peanut allergic donor peanutspecific Ara h 2 pulsed TCL cytokine responses to Ara h 2 peptides, Arah 2 and crude peanut extract. Peanut specific TCLs (5×10⁴/ml) fromindividuals with peanut allergy were generated using CPE, thenstimulated with Ara h 2 peptides at a concentration of 10 ug/ml in thepresence of autologous irradiated PBMC as APC (5×10⁴/ml) in 3 daycultures. Cytokine supernatants were collected after 48 hours cultureand assayed by specific ELISA. Data is displayed graphically asIL-5:IFN-γ ratio. Peptides associated with a significant proliferativeresponse are shaded grey.

FIG. 8 is a graphical representation of peanut allergic donor Ara h 2pulsed TCL cytokine responses to Ara h 2 peptides, Ara h 2 and crudepeanut extract. Peanut specific TCL (5×10⁴ cells/ml) from individualswith peanut allergy generated using CPE and pulsed with rAra h 2, werestimulated with Ara h 2 peptides at a concentration of 10 μg/ml in thepresence of autologous irradiated PBMC as APC (5×10⁴ cells/ml) in 3 daycultures. Cytokine supernatants were collected after 48 hours cultureand assayed by specific ELISA. Data are displayed graphically as IL-5concentration (pg/ml). Peptides associated with a significantproliferative response are shaded grey.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the identification ofAra h 2 T cell epitopic regions. The identification of immunodominantepitopes of Ara h 2 has enabled the improvement of diagnosticmethodology and the development of therapeutic and prophylacticcompositions and treatment approaches for conditions such as, but notlimited to, peanut allergy. In accordance with the present invention,overlapping peptides were synthesised based on the Ara h 2 amino acidsequence disclosed in SEQ ID NO:1. The T cell immunoreactivity of thesepeptides is identified in accordance with the present invention on thebasis of the interactivity of T cell lines generated from the peripheralblood of subjects with peanut allergies. The identification andgeneration of those molecules thereby forms the basis for a new range ofdiagnostic, therapeutic and prophylactic reagents and procedures.

Accordingly, one aspect of the present invention provides an isolatedpeptide of the formula:X₁X₂X₃

wherein:

-   -   X₁ and X₃ may be the same or different and each is an amino acid        sequence comprising from 0 to 40 naturally or non-naturally        occurring amino acid residues;    -   X₂ is any amino acid sequence derived from or homologous to Ara        h 2;

and wherein said peptide molecule is capable of interacting with T cellsand modifying T cell function when incubated with cells from subjectshaving a condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,analogue, mutant, chemical equivalent or mimetic of said peptide.

Without limiting the present invention in any way, peanuts contain manyproteins, with the number of distinct bands visible on SDS-PAGEdepending on the methodology used. Up to 53 bands are visible followinghigh pressure liquid chromatography (de Jong et al., Clin Exp Allergy28: 743-51, 1998). Only two of these proteins warrant classification asmajor allergens using standard criteria, whereby IgE reactivity occurswithin greater than 50% of the peanut allergic population; theseproteins are termed Ara h 1 and Ara h 2 (Burks et al., Allergy 53:725-30, 1998).

Ara h 2 is a glycoprotein which has been identified as a member of theconglutin seed storage family. 20% of the Ara h 2 molecular massrepresents carbohydrate side chains and it migrates as a doublet onSDS-PAGE with an average molecular mass of 17.5 kDa (Burks et al, IntArch Allergy Immunol 119:165-172, 1992). It has been characterised as amajor allergen, on the basis of its reactivity with 6 out of 6 seratested (Burks et al, 1992, supra). Others have also confirmed itsimportance Clarke demonstrated that 71% of subjects possessed specificIgE to Ara h 2 upon western blotting of crude peanut extract.Kleber-Janke et al. have demonstrated that 85% of subjects possessed IgEtowards their recombinant form upon western blotting, and de Jong'sgroup have shown that approximately 78% of their group demonstratespecific IgE to purified natural Ara h 2 (Clarke et al., Clin ExpAllergy 28: 1251-7, 1998; de Jong et al, 1998 supra; Kleber-Janke etal., Int Arch Allergy Immunol 119: 265-274, 1999). Linear epitopemapping has demonstrated 10 IgE binding epitopes throughout Ara h 2,with 3 potentially immunodominant.

Reference to “Ara h 2” should be understood to include reference to allforms and components of Ara h 2 or derivatives, mutants, homologues,analogues, equivalents or mimetics thereof. Reference to “Ara h 2”should also be understood to include reference to all protein forms ofAra h 2 or its functional equivalent or derivative including, forexample, any isoforms which may arise from alternative splicing of Ara h2 mRNA. It includes reference to mutants, polymorphic variants orhomologues of Ara h 2. It also includes reference to analogues orequivalents of Ara h 2 such as may occur where a product which naturallycomprises Ara h 2 is synthetically generated for the purpose ofgenerating a product such as a food additive. The present inventionthereby provides epitopes and methods for their use in the diagnosis andtreatment of any condition characterised by hypersensitivity to an Ara h2 or Ara h 2-like molecule such as peanut allergy or a tree-nut allergy.Preferably, said Ara h 2 comprises the sequence set forth in SEQ ID NO:1or is a derivative, homologue, analogue, chemical equivalent, mutant ormimetic of said sequence.

The present invention therefore more particularly provides an isolatedpeptide of the formula:X₁X₂X₃

wherein:

-   -   X₁ and X₃ may be the same or different and each is an amino acid        sequence comprising from 0 to 40 naturally or non-naturally        occurring amino acid residues;    -   X₂ is an amino acid sequence of from 5 to 100 residues derived        from, homologous to or contiguous with amino acids 1-157        inclusive or derivatives thereof of Ara h 2;

and wherein said peptide molecule is capable of interacting with T cellsand modifying T cell function when incubated with cells from subjectshaving a condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,mutant, chemical equivalent or mimetic of said peptide.

Still more particularly the present invention provides an isolatedpeptide of the formula:X₁X₂X₃

wherein

-   -   X₁ and X₃ may be the same or different and each is an amino acid        sequence comprising from 0 to 40 naturally or non-naturally        occurring amino acid residues;    -   X₂ is an amino acid sequence of from 5 to 100 residues derived        from, homologous to or contiguous with amino acids 19-92, 91-137        and/or 127-155 inclusive or derivatives thereof of Ara h 2;

and wherein said peptide molecule is capable of interacting with T cellsand modifying T cell function when incubated with cells from subjectshaving a condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,analogue, mutant, chemical equivalent or mimetic of said peptide.

Still more particularly, X₂ is any amino acid sequence of from 5 to 100residues derived from, homologous to or contiguous with amino acids19-38, 28-47, 37-56, 46-65, 55-74, 73-92, 82-101, 91-110, 100-119,118-137, 127-146 and/or 136-155 inclusive or derivatives thereof of Arah 2.

Yet more particularly, X₂ is any amino acid sequence of from 5 to 100residues derived from homologous to or contiguous with amino acids19-38, 28-47, 73-92, 91-110 and/or 100-119 inclusive or derivativesthereof of Ara h 2.

Most particularly, X₂ is any amino acid sequence of from 5 to 100residues derived from, homologous to or contiguous with amino acids19-38, 28-47, 73-92 and/or 100-119 inclusive or derivatives thereof ofAra h 2.

Reference to “T cells” should be understood as a reference to any cellcomprising a T cell receptor. In this regard, the T cell receptor maycomprise any one or more of the α, β, γ or δ chains. The presentinvention is not intended to be limited to any particular functionalsub-class of T cells although in a preferred embodiment the subject Tcell is a T helper cell and still more preferably a Th2-type cell. Inthis regard, reference to “modifying T cell function” should beunderstood as a reference to modifying any one or more functions which aT cell is capable of performing. For example, the subject function maybe proliferation, differentiation or other form of cellular functionalactivity such as the production of cytokines. Preferably, the subjectfunctional activity is proliferation.

In terms of modifying the function of T cells from subjects having acondition characterised by an aberrant, unwanted or inappropriate immuneresponse to Ara h 2, it should be understood that this is notnecessarily a reference to modifying the function of all the T cells ina given sample but is likely, in fact, to reflect the modification orfunctioning of only some of the T cells in the sample. For example, onlya portion of the T helper cells in a given T cell sample mayfunctionally respond to contact with the subject peptide. Such a partialresponse should be understood to fall within the scope of the presentinvention. It should also be understood that the T cells which arederived from the subject may be freshly harvested T cells or they mayhave undergone some form of in vitro or in vivo manipulation prior totesting. For example, T cell lines may have been generated from the cellsample and it is these T cell lines which then form the subject derivedT cell population which is tested in accordance with the presentinvention. To the extent that the subject functional activity is T cellproliferation, the T cell proliferation assay is preferably performed asdisclosed herein. Still more preferably, the subject modification of Tcell function is the induction of a proliferation index of ≧2.5.

Reference to an “aberrant, unwanted or otherwise inappropriate” immuneresponse should be understood as a reference to any form ofphysiological activity which involves the activation and/or functioningof one or more immune cells where that activity is inappropriate in thatit is of an inappropriate type or proceeds to an inappropriate degree.It may be aberrant in that according to known immunological principalsit either should not occur when it does so or else should occur when itdoes not do so. In another example, the immune response may beinappropriate in that it is a physiologically normal response but whichis unnecessary and/or unwanted, such as occurs with respect to type-Ihypersensitivity responses to innocuous allergens. Preferably saidimmune response is peanut hypersensitivity.

By “peanut hypersensitivity” it should be understood to mean theexhibition of clinical symptoms of IgE mediated peanut hypersensitivity.However, it should be understood that although clinical symptoms may beevident, not all such individuals would necessarily exhibit detectablelevels of peanut specific serum IgE which is measured using theKallestad Allercoat EAST System (Sanofi-Pasteur Diagnostics, USA),although such individuals should nevertheless be understood to fallwithin the scope of the definition of those exhibiting “peanuthypersensitivity”. Alternatively, testing may proceed utilising eitherthe Pharmacia or the UniCap systems.

In a preferred embodiment, X₂ comprises not less than about 5 and notgreater than about 50 amino acid residues, more preferably not less thanabout 5 and not greater than about 30 amino acid residues and even morepreferably not less than about 5 and not greater than about 20. Mostpreferably, X₂ comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 amino acids.

In a particularly preferred embodiment, X₂ comprises a sequence of atleast 5 amino acids derived from one or more of the following amino acidsequences: RQQWELQGDRRCQSQLERAN (SEQ ID NO:4) RRCQSQLERANLRPCEQHLM (SEQID NO:5) PYDRRGAGSSQHQERCCNEL (SEQ ID NO:10) RCMCEALQQIMENQSDRLQG (SEQID NO:13)

More preferably, X₂ comprises a sequence of at least 5 amino acidsderived from one or more of SEQ ID NO:4 or SEQ ID NO:5.

Reference to a “peptide” includes reference to a peptide, polypeptide orprotein or parts thereof. The peptide may be glycosylated orunglycosylated and/or may contain a range of other molecules fused,linked, bound or otherwise associated to the protein such as aminoacids, lipids, carbohydrates or other peptides, polypeptides orproteins. Reference hereinafter to a “peptide” includes a peptidecomprising a sequence of amino acids as well as a peptide associatedwith other molecules such as amino acids, lipids, carbohydrates or otherpeptides, polypeptides or proteins.

“Derivatives” include fragments, parts, portions and variants fromnatural, synthetic or recombinant sources including fusion proteins.Parts or fragments include, for example, active regions of the subjectpeptide. Derivatives may be derived from insertion, deletion orsubstitution of amino acids. Amino acid insertional derivatives includeamino and/or carboxylic terminal fusions as well as intrasequenceinsertions of single or multiple amino acids. Insertional amino acidsequence variants are those in which one or more amino acid residues areintroduced into a predetermined site in the protein although randominsertion is also possible with suitable screening of the resultingproduct. Deletional variants are characterized by the removal of one ormore amino acids from the sequence.

Substitutional amino acid variants are those in which at least oneresidue in the sequence has been removed and a different residueinserted in its place. An example of substitutional amino acid variantsare conservative amino acid substitutions. Conservative amino acidsubstitutions typically include substitutions within the followinggroups: glycine and alanine; valine, isoleucine and leucine; asparticacid and glutamic acid; asparagine and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine. Additions to aminoacid sequences include fusions with other peptides, polypeptides orproteins.

Chemical and functional equivalents of the subject peptide should beunderstood as molecules exhibiting any one or more of the functionalactivities of these molecules and may be derived from any source such asbeing chemically synthesized or identified via screening processes suchas natural product screening.

Homologues include peptides derived from varieties other than peanuts,such as peptides derived from other tree nuts.

The derivatives include fragments having particular epitopes or parts ofthe entire protein fused to peptides, polypeptides or otherproteinaceous or non-proteinaceous molecules.

Analogues contemplated herein include, but are not limited to,modification to side chains, incorporating of unnatural amino acidsand/or their derivatives during peptide, polypeptide or proteinsynthesis and the use of crosslinkers and other methods which imposeconformational constraints on the proteinaceous molecules or theiranalogues. Mutants include molecules which exhibit modified functionalactivity (for example, Ara h 2 peptides which express one or more T cellepitopes but lack B cell reactivity).

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivatisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringprotein synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acids contemplated herein is shown in Table 2. TABLE 2Non-conventional Non-conventional amino acid Code amino acid Codeα-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrateMgabu L-N-methylarginine Nmarg aminocyclopropane- CproL-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmaspaminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- NorbL-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglucyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanineCpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine NmleuD-arginine Darg L-N-methyllysine Nmlys D-aspartic acid DaspL-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine NmnleD-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid DgluL-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine NmpheD-isoleucine Dile L-N-methylproline Nmpro D-leucine DleuL-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine NmthrD-methionine Dmet L-N-methyltryptophan Nmtrp D-ornithine DornL-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline NmvalD-proline Dpro L-N-methylethylglycine Nmetg D-serine DserL-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine NleD-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyra-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl-Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH2)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety.

It is possible to modify the structure of a peptide according to theinvention for various purposes such as for increasing solubility,enhancing therapeutic or preventative efficacy, enhancing stability orincreasing resistance to proteolytic degradation. A modified peptide maybe produced in which the amino acid sequence has been altered, such asby amino acid substitution, deletion or addition, to modifyimmunogenicity and/or reduce allergenicity. Similarly components may beadded to peptides of the invention to produce the same result.

For example, a peptide can be modified so that it exhibits the abilityto induce T cell anergy. In this instance, critical binding residues forthe T cell receptor can be determined using known techniques (forexample substitution of each residue and determination of the presenceor absence of T cell reactivity) In one example, those residues shown tobe essential to interact with the T cell receptor can be modified byreplacing the essential amino acid with another, preferably similaramino acid residue (a conservative substitution) whose presence is shownto alter T cell reactivity or T cell functioning. In addition, thoseamino acid residues which are not essential for T cell receptorinteraction can be modified by being replaced by another amino acidwhose incorporation may then alter T cell reactivity or T cellfunctioning but does not, for example, eliminate binding to relevant MHCproteins. In yet another example, mutant peptides may be created whichexhibit normal T cell binding but abrogated IgE binding.

Exemplary conservative substitutions are detailed in Table 3, below, andinclude: Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln IleLeu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, TyrSer Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Such modifications will result in the production of molecules fallingwithin the scope of “mutants” of the subject peptide as herein defined.“Mutants” should be understood as a reference to peptides which exhibitone or more structural features or functional activities which aredistinct from those exhibited by the non-mutated peptide counterpart.Peptides of the invention may also be modified to incorporate one ormore polymorphisms resulting from natural alielic variation and D-aminoacids, non-natural amino acids or amino acid analogues may besubstituted into the peptides to produce modified peptides which fallwithin the scope of the invention. Peptides may also be modified byconjugation with polyethylene glycol (PEG) by known techniques. Reportergroups may also be added to facilitate purification and potentiallyincrease solubility of the peptides according to the invention. Otherwell known types of modification including insertion of specificendoprotease cleavage sites, addition of functional groups orreplacement of hydrophobic residues with less hydrophobic residues aswell as site-directed mutagenesis of DNA encoding the peptides of theinvention may also be used to introduce modifications which could beuseful for a wide range of purposes. The various modifications topeptides according to the invention which have been mentioned above arementioned by way of example only and are merely intended to beindicative of the broad range of modifications which can be effected.

In related aspects, the method of the present invention provides amutant form of the peptides hereinbefore defined wherein said peptidemolecule retains all or some of its capacity to interact with T cellsbut exhibits partially or completely inhibited, abrogated or otherwisedown-regulated antibody reactivity. Effecting the down-regulation ofantibody reactivity can be achieved by any suitable method, whichmethods would be well known to those skilled in the art. For example, tothe extent that a B cell epitope is defined by its linear amino acidsequence, one may add, delete or substitute one or more amino acidresidues in order to render the mutated linear sequence distinct fromthe naturally occurring sequence. To the extent that an epitope may beadditionally, or alternatively, defined by a conformational epitope, onemay seek to disrupt that conformation by disrupting a 2° or, to theextent that homodimers or heterodimers exist, a 3° structure of thepeptide. This may be achieved, for example, by disrupting the formationof bonds, such as disulphide bonds, which are known to stabilise 2°and/or 3° structures.

Accordingly, in a related aspect the present invention provides apeptide, as hereinbefore defined, wherein the antibody reactivity ofsaid peptide is inhibited, abrogated or otherwise down-regulated.

More preferably, said antibody is IgE.

Another aspect of the present invention provides an isolated peptidecomprising any amino acid sequence derived from or homologues to Ara h 2wherein said peptide molecule is capable of interacting with T cells andmodifying T cell function when incubated with cells from subjects havinga condition characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or a derivative, homologue,analogue, mutant, chemical equivalent or mimetic of said peptide.

More particularly, the present invention provides an isolated peptidecomprising an amino acid sequence of from 5-100 residues derived from,homologues to or contiguous with amino acids 1-157 inclusive orderivatives thereof of Ara h 2 wherein said peptide molecule is capableof interacting with T cells and modifying T cell function when incubatedwith cells from subjects having a condition characterised by anaberrant, unwanted or otherwise inappropriate immune response to Ara h 2or a derivative, homologue, analogue, mutant, chemical equivalent ormimetic of said peptide.

In one preferred embodiment said amino acid sequence is derived from,homologous to or contiguous with amino acids 1-92, 91-137 and/or 127-155inclusive or derivatives thereof of Ara h 2.

In another preferred embodiment said amino acid sequence is derivedfrom, homologous to or contiguous with amino acids 19-38, 28-47, 37-56,46-65, 55-74, 73-92, 82-101, 91-110, 100-119, 118-137, 127-146 and/or136-155 inclusive or derivatives thereof of Ara h 2.

In yet another preferred embodiment said amino acid sequence is derivedfrom, homologous to or contiguous with amino acids 19-38, 28-47, 73-92,91-110 and/or 100-119 inclusive or derivatives thereof of Ara h 2.

More preferably, said amino acid sequence is derived from, homologous toor contiguous with amino acids 1-38, 28-47, 73-92 and/or 100-119inclusive or derivatives thereof of Ara h 2.

In another aspect said amino acid sequence comprises a sequence of atleast 5 amino acids derived from one or more of the following amino acidsequences: RQQWELQGDRRCQSQLERAN (SEQ ID NO:4) RRCQSQLERANLRPCEQHLM (SEQID NO:5) PYDRRGAGSSQHQERCCNEL (SEQ ID NO:10) RCMCEALQQIMENQSDRLQG (SEQID NO:13)

According to this aspect, said amino acid sequence preferably comprisesa sequence of at least 5 amino acids derived from one or more of SEQ IDNO:4 or SEQ ID NO:5.

The peptides of the present invention may be prepared by recombinant orchemical synthetic means. According to a preferred aspect of the presentinvention, there is provided a recombinant peptide or mutant thereofwhich is preferentially immunologically reactive with T cells fromindividuals with peanut hypersensitivity, which is expressed by theexpression of a host cell transformed with a vector coding for thepeptide sequence of the present invention. The peptide may be fused toanother peptide, polypeptide or protein. Alternatively, the peptide maybe prepared by chemical synthetic techniques, such as by the Merrifieldsolid phase synthesis procedure. Furthermore, although syntheticpeptides of the formula given above represent a preferred embodiment,the present invention also extends to biologically pure preparations ofthe naturally occurring peptides or fragments thereof. By “biologicallypure” is meant a preparation comprising at least about 60%, preferablyat least about 70%, or preferably at least about 80% and still morepreferably at least about 90% or greater as determined by weight,activity or other suitable means.

The present invention should therefore be understood to encompasspeptides that comprise at least one T or B cell epitope of Ara h 2 inconjunction with other amino acids (which may or may not be naturallyoccurring as amino acid analogues) or other chemical species. In apreferred aspect of the invention such peptides may comprise one or moreepitopes of Ara h 2, which epitopes may be T or B cell epitopes.Peptides with one or more epitopes of Ara h 2 are desirable forincreased therapeutic effectiveness.

In another aspect, the present invention provides a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding the peptides as hereinbefore defined or aderivative, homologue or analogue thereof.

It should be understood that reference to “peptides” includes referenceto peptides comprising one or more T cell epitopes. A nucleic acidmolecule encoding the subject peptide is preferably a sequence ofdeoxyribonucleic acids such as cDNA or a genomic sequence. A genomicsequence may comprise exons and introns. A genomic sequence may alsoinclude a promoter region or other regulatory regions.

The nucleic acid molecule may be ligated to an expression vector capableof expression in a prokaryotic cell (eg. E. coli) or a eukaryotic cell(eg. yeast cells, fungal cells, insect cells, mammalian cells or plantcells). The nucleic acid molecule may be ligated or fused or otherwiseassociated with a nucleic acid molecule encoding another entity such as,for example, a signal peptide. It may also comprise additionalnucleotide sequence information fused, linked or otherwise associatedwith it either at the 3′ or 5′ terminal portions or at both the 3′ and5′ terminal portions. The nucleic acid molecule may also be part of avector, such as an expression vector. The latter embodiment facilitatesproduction of recombinant forms of the subject peptide which forms areencompassed by the present invention.

Such nucleic acids may be useful for recombinant production of T cellepitopes of Ara h 2 or proteins comprising them by insertion into anappropriate vector and transfection into a suitable cell line. Suchexpression vectors and host cell lines also form an aspect of theinvention.

In producing peptides by recombinant techniques, host cells transformedwith a nucleic acid having a sequence encoding a peptide according tothe invention or a functional equivalent of the nucleic acid sequenceare cultured in a medium suitable for the particular cells concerned.Peptides can then be purified from cell culture medium, the host cellsor both using techniques well known in the art such as ion exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis or immunopurification with antibodies specific for thepeptide.

Nucleic acids encoding Ara h 2 or peptides comprising T and/or B cellepitopes of Ara h 2 may be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells such as Chinese hamster ovarycells (CHO). Suitable expression vectors, promoters, enhancers and otherexpression control elements are referred to in Sambruck et al (1989).Other suitable expression vectors, promoters, enhancers and otherexpression elements are well known to those skilled in the art. Examplesof suitable expression vectors in yeast include Yep Sec 1 (Balderi etal., 1987, Embo J., 6.229-234); pMFa (Kurjan and Herskowitz., 1982,Cell., 30:933-943); JRY88 (Schultz et al., 1987, Gene., 54.113-123) andpYES2 (Invitrogen Corporation, San Diego, Calif.). These vectors arefreely available as are baculovirus and mammalian expression systems.For example, a baculovirus system is commercially available (ParMingen,San Diego, Calif.) for expression in insect cells while the pMsg vectoris commercially available (Pharmacia, Piscataway, N.J.) for expressionin mammalian cells.

For expression in E. coli suitable expression vectors include amongothers, pTrc (Amann et al., 1998, Gene., 69.:301-315) pGex (AmradCorporation, Melbourne, Australia); pMal (N.E. Biolabs, Beverley,Mass.); pRit5 (Pharmacia, , Piscataway, N.J.); pEt-11d (Novagen,Maddison, Wis.) (Jameel et al., 1990, J. Virol., 64.3963-3966) and pSem(Knapp et al., 1990, Bio Techniques., 8.280-281). The use of pTRC, andpEt-11d, for example, will lead to the expression of unfused protein.The use of pMal, pRit5, pSem and pGex will lead to the expression ofallergen fused to maltose E binding protein (pMal), protein A (pRit5),truncated-galactosidase (PSEM) or glutathione S-transferase (pGex). Whena T cell epitope of Ara h 2 or a peptide comprising it is expressed as afusion protein, it is particularly advantageous to introduce anenzymatic cleavage site at the fusion junction between the carrierprotein and the peptide concerned. The peptide of the invention may thenbe recovered from the fusion protein through enzymatic cleavage at theenzymatic site and biochemical purification using conventionaltechniques for purification of proteins and peptides. The differentvectors also have different promoter regions allowing constitutive orinducible expression or temperature induction. It may additionally beappropriate to express recombinant peptides in different E. coli hoststhat have an altered capacity to degrade recombinantly expressedproteins. Alternatively, it may be advantageous to alter the nucleicacid sequence to use codons preferentially utilised by E. coli, wheresuch nucleic acid alteration would not effect the amino acid sequence ofthe expressed proteins.

Host cells can be transformed to express the nucleic acids of theinvention using conventional techniques such as calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection orelectroporation. Suitable methods for transforming the host cells may befound in Sambruck et al. (1989), and other laboratory texts. The nucleicacid sequence of the invention may also be chemically synthesised usingstandard techniques.

In addition to recombinant production of peptides according to theinvention, the nucleic acids may be utilised as probes for experimentalor purification purposes.

The identification of T cell epitopic regions facilitates theidentification and/or rational design of a range of mutant peptidemolecules. As detailed hereinbefore, these mutant peptides may compriseone or more mutated T cell epitopes and/or B cell epitopes. In thisregard, there is provided scope for the generation of mutant peptidescomprising mutated B cell epitopes or combinations of intact versusmutated B and T cell epitopes. The applications of these molecules aredescribed in more detail below but in a preferred embodiment relate tomodulation of the peanut hypersensitivity immune response in terms ofeither a prophylactic or therapeutic treatment.

Identification and synthesis of the Ara h 2 T cell epitopes as disclosedherein now facilitates the development of a range of diagnostic andprophylactic/therapeutic treatment protocols for use with respect topeanut related immune conditions. Also facilitated is the development ofreagents for use therein. Accordingly, the present invention should beunderstood to extend to the use of the peptides or functionalderivatives, homologues, analogues or mutants thereof in the therapeuticand/or prophylactic treatment of patients. Such methods of treatmentinclude, but are not limited to:

-   -   (i) Administration of the subject peptides or mutants thereof to        a patient as a means of desensitising or inducing immunological        tolerance to Ara h 2 or Ara h 2-like molecules. This may be        achieved, for example, by inducing Ara h 2 directed Th2 anergy        or apoptosis. Such an outcome may be achieved by any one of a        number of techniques including the use of peptides which        maintain T cell epitope reactivity but which either naturally or        as a result of mutation are unable to undergo IgE binding.        Alternatively, one may utilise desensitisation/treatment        protocols which are based on the administration of specific        concentrations of a given peptide in accordance with a specific        regime in order to induce tolerance. Such methodology may        eliminate Ara h 2 hypersensitivity or it may reduce the severity        of Ara h 2 hypersensitivity.        -   Preferably such treatment regimes are capable of modifying            the T cell response or both the B and T cell response of the            individual concerned. As used herein, modification of the            allergic response of the individual suffering from peanut            hypersensitivity can be defined as inducing either            non-responsiveness or diminution in symptoms to the Ara h 2            molecule as determined by standard clinical procedures            (Varney et al. 1991 British Medical Journal 302:265-269).            Diminution in the symptoms includes any reduction in an            allergic response in an individual to Ara h 2 after a            treatment regime has been completed. This diminution may be            subjective or clinically determined, for example by using            standard skin tests known in the art.        -   Exposure of an individual to the peptides of the present            invention, which peptides comprise at least one T cell            epitope, may tolerise or anergise appropriate T cell            subpopulations such that they become unresponsive to Ara h 2            and do not participate in stimulating an immune response            upon such exposure. Preferably the peptides according to the            invention will retain immunodominant T cell epitopes but            possess abrogated IgE binding.        -   Administration of a peptide of the invention may modify the            cytokine secretion profile as compared with exposure to            naturally occurring Ara h 2 allergen. This exposure may also            influence T cell subpopulations which normally participate            in the allergic response to migrate away from the site or            sites of normal exposure to the allergen and towards the            site or sites of therapeutic administration. This            redistribution of T cell subpopulations may ameliorate or            reduce the ability of an individual's immune system to            stimulate the usual immune response at the site of normal            exposure to the allergen, resulting in diminution of the            allergic symptoms.        -   Modification of the B cell response may be achieved, for            example, via modulation of the cytokine profile produced by            T cells, as detailed above. Specifically, decreasing T cell            derived IL-4 and IL-1 3 production thereby decreasing IgE            synthesis.    -   (ii) The peptides of the present invention may be used in the        capacity of an adsorbent to remove Ara h 2 directed T cells from        a biological sample or from a patient.

Accordingly, in another aspect the present invention provides a methodfor the treatment and/or prophylaxis of a condition in a subject, whichcondition is characterised by the aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2, said method comprisingadministering to said subject an effective amount of a peptide ashereinbefore defined for a time and under conditions sufficient toremove or reduce the presence or function in said subject of T cellsdirected to said Ara h 2.

Preferably said condition is hypersensitivity to peanuts or tree nutswhich contain Ara h 2 or Ara h 2-like molecules, such as hazelnuts,almonds or Brazil nuts.

An “effective amount” means an amount necessary at least partly toattain the desired immune response, or to delay the onset or inhibitprogression or halt altogether, the onset or progression of a particularcondition being treated. The amount varies depending upon the health andphysical condition of the individual to be treated, the taxonomic groupof individual to be treated, the degree of protection desired, theformulation of the composition, the assessment of the medical situation,and other relevant factors. It is expected that the amount will fall ina relatively broad range that can be determined through routine trials.

The subject of the treatment or prophylaxis is generally a mammal suchas but not limited to human, primate, livestock animal (e.g. sheep, cow,horse, donkey, pig), companion animal (e.g. dog, cat), laboratory testanimal (e.g. mouse, rabbit, rat, guinea pig, hamster), captive wildanimal (e.g. fox, deer). Preferably the mammal is a human or primate.Most preferably the mammal is a human.

Reference herein to “treatment” and “prophylaxis” is to be considered inits broadest context. The term “treatment” does not necessarily implythat a subject is treated until total recovery. Similarly, “prophylaxis”does not necessarily mean that the subject will not eventually contracta disease condition. Accordingly, treatment and prophylaxis includeamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylaxis” may be considered as reducing the severity or onsetof a particular condition. “Treatment” may also reduce the severity ofan existing condition.

Administration of the peptide of the present invention (herein referredto as “agent”) in the form of a pharmaceutical composition, may beperformed by any convenient means. The agent of the pharmaceuticalcomposition is contemplated to exhibit therapeutic activity whenadministered in an amount which depends on the particular case. Thevariation depends, for example, on the human or animal and the agentchosen. A broad range of doses may be applicable. Considering a patient,for example, from about 0.1 mg to about 1 mg of an agent may beadministered per kilogram of body weight per day. Dosage regimes may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily, weekly, monthly orother suitable time intervals or the dose may be proportionally reducedas indicated by the exigencies of the situation.

The agent may be administered in a convenient manner such as by theoral, intravenous (where water soluble), intraperitoneal, intramuscular,subcutaneous, intradermal, intranasal, sublingual or suppository routesor implanting (e.g. using slow release molecules). The agent may beadministered in the form of pharmaceutically acceptable nontoxic salts,such as acid addition salts or metal complexes, e.g. with zinc, iron orthe like (which are considered as salts for purposes of thisapplication). Illustrative of such acid addition salts arehydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate,citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.If the active ingredient is to be administered in tablet form, thetablet may contain a binder such as tragacanth, corn starch or gelatin;a disintegrating agent, such as alginic acid; and a lubricant, such asmagnesium stearate.

In accordance with these methods, the agent defined in accordance withthe present invention may be coadministered with one or more othercompounds or molecules. By “coadministered” is meant simultaneousadministration in the same formulation or in two different formulationsvia the same or different routes or sequential administration by thesame or different routes. By “sequential” administration is meant a timedifference of from seconds, minutes, hours or days between theadministration of the two types of molecules. These molecules may beadministered in any order.

Another aspect of the present invention contemplates the use of an agentas hereinbefore defined in the manufacture of a medicament for thetreatment of a condition in a mammal, which condition is characterisedby an aberrant, unwanted or otherwise inappropriate immune response toAra h 2.

Preferably said condition is hypersensitivity to peanuts or a tree nutwhich contains Ara h 2 or Ara h 2-like molecules, such as a hazelnut.

In yet another further aspect, the present invention contemplates apharmaceutical composition comprising an agent as hereinbefore definedand one or more pharmaceutically acceptable carriers and/or diluents.Said agents are referred to as the active ingredients.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion or may be in the form of a cream or other formsuitable for topical application. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsuperfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the various sterilised active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active ingredients are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in suchtherapeutically useful compositions in such that a suitable dosage willbe obtained. Preferred compositions or preparations according to thepresent invention are prepared so that an oral dosage unit form containsbetween about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter: a binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen, or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound(s) may be incorporated intosustained-release preparations and formulations.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule encoding a modulatory agent. The vectormay, for example, be a viral vector.

Routes of administration include, but are not limited to, respiratorally(eg. intranasally or orally via aerosol), intratracheally,nasopharyngeally, intravenously, intraperitoneally, subcutaneously,intracranially, intradermally, intramuscularly, intraoccularly,intrathecally, intracereberally, intranasally, infusion, orally,rectally, via IV drip patch and implant. Preferably, said route ofadministration is subcutaneously, intradermally or intranasally.

Yet another aspect of the present invention relates to agents, ashereinbefore defined, when used in the method of the present invention.

In yet another aspect, the present invention should be understood toextend to the use of the peptides of the present invention in diagnosticapplications. Said diagnostic applications include, but are not limitedto:

-   -   (i) To measure the reactivity of a subject's cells to Ara h 2.        This is of use, for example, with respect to the diagnosis        and/or monitoring of conditions characterised by an aberrant,        unwanted or otherwise inappropriate immune response to Ara h 2.        The peptides may be added into solution or bound to a solid        support together with cells derived from peripheral blood or        from tissue biopsies either unfractionated, fractionated or        derived as a continuous cell line. Reactivity to the subject        peptide may then be measured by standard proliferation assays        such as incorporation of H³-thymidine, measurement of expressed        or secreted molecules such as surface markers, cytokines or        other standard assays of cellular activity which are well known        in the art.    -   (ii) The use of T cell epitope comprising peptides together with        a T cell proliferation assay which utilises a T cell sample        derived from the subject will facilitate, for example, the        identification of a T cell responsive population.

Methods of detecting Ara h 2 may be utilised, for example, toqualitatively or quantitatively detect Ara h 2 levels. However, thesemethods may also be utilised to screen for mutations or polymorphisms inAra h 2 which mutations may result in, for example, loss of T cellreactivity to Ara h 2. These methods may be utilised for the purpose ofscreening for peptide molecules suitable for use in therapeutically orprophylactically treating an individual suffering from Ara h 2 relatedhypersensitivity.

Accordingly, yet another aspect of the present invention is directed toa method of diagnosing or monitoring a condition in a mammal, whichcondition is characterised by an aberrant, unwanted or inappropriateresponse to Ara h 2, said method comprising screening for Ara h 2reactive T cells utilising the peptides hereinbefore defined.

Preferably said condition is hypersensitivity to peanuts or tree nutswhich contain Ara h 2 or Ara h 2-like molecules, such as hazelnuts,almonds or Brazil nuts.

In another embodiment the present invention provides diagnostic kits foruse in the diagnostic methodology hereinbefore defined.

The present invention will now be further described with reference tothe following non-limiting Examples.

EXAMPLE 1 Materials & Methods

The materials and methods detailed in this example were utilised inrelation to the Example 2 data. 1. Materials

Tissue Culture Reagents

Ara h 2 20-mer peptides Chiron Mimotopes, Australia

Ficoll-Paque (research grade) Amersham Biosciences, Sweden

Foetal Bovine Serum (FBS) Gibco BRL, USA

Human AB+ serum Sigma Chemical Company, USA

Penicillin-Streptomycin-Glutamine Gibco BRL, USA

Phytohaemagglutinin (PHA) Wellcome Diagnostics, England

Recombinant human IL-2 (rIL-2) Cetus Corporation, USA

RPMI 1640 (glutamine free) Gibco BRL, USA

Sodium heparin (preservative free) David Bull Laboratories, Australia

Tetanus toxoid Laboratory stocks

Cell Lines and Bacterial Transfectants

rAra h 2 transfected BL-21 E. coli Kind gift of Ms M de Leon

Immunoblotting Reagents

Nitrocellulose membrane (BA 0.45 μm) Schleicher and Schuell, Germany

Rabbit anti-mouse/human immunoglobulin- Dako Corporation, Denmarkhorseradish peroxidase (HRPO) conjugate

Skim milk powder Diploma, Australia

Ponceau S Sigma Chemical Company, USA

Enhanced chemiluminescence reagent Pierce, USA

ELISA Reagents

Biotinylated rat-anti-human IFN-γ Endogen, USA

Biotinylated rat-anti-human IL-5 PharMingen, USA

Human recombinant IFN-γ Endogen, USA

Human recombinant IL-5 PharMingen, USA

Mouse anti-human IFN-γ Endogen, USA

Mouse anti-human IL-5 PharMingen, USA

Streptavidin-biotinylated HRPO complex Amersham, USA

Enhanced chemiluminescence reagent Perkin Elmer, USA

Flow Cytometry Reagents

Mouse anti-human CD4-FITC PharMingen, USA

Mouse anti-human CD4-FITC/CD8-PE Becton Dickinson, USA

Mouse anti-human CD3-FITC/CD19-PE Becton Dickinson, USA

Mouse anti-human CD45-FITC/CD14-PE Becton Dickinson, USA

Mouse IgG₁-FITC/IgG₁-PE isotype control Becton Dickinson, USA

Mouse IgG₁-APC isotype control PharMingen, USA

Mouse IgG₁-FITC isotype control PharMingen, USA

Mouse IgG₁-PE isotype control PharMingen, USA

Phorbol 12-myristate 13-acetate (PMA) Sigma Chemical Company, USA

General Reagents

Dialysis tubing (MW cut off 3.5 kDa) Pierce, USA General Chemicals

Ammonium persulphate (AP) Bio-Rad Laboratories, USA

Ampicillin Sigma Chemical Company, USA

Aqueous counting scintillant (ASCII) Ajax Chemicals, Australia

Bis-acrylamide, electrophoresis purity Bio-Rad Laboratories, USA

Bovine Serum Albumin (BSA) Sigma Chemical Company, USA

Bovine γ-globulin (BGG) Sigma Chemical Company, USA

Bromophenol blue BDH Laboratory Supplies, UK

Coomassie Brilliant Blue R-250 Bio-Rad Laboratories, USA

Dimethylpimelimidate (DMP) Sigma Chemical Company, USA

Dimethylsulphoxide (DMSO) Sigma Chemical Company, USA

Dithiothreitol (DTT) Sigma Chemical Company, USA

Ethanol, 95% (absolute) Ajax Chemicals, Australia

Glacial acetic acid Ajax Chemicals, Australia

Glycerol Ajax Chemicals, Australia

Glycine Ajax Chemicals, Australia

Hydrogen chloride Ajax Chemicals, Australia

Hydrogen peroxide (H₂O₂) (30%) Ajax Chemicals, Australia

Hydrogen sulphate (H₂SO₄), analytical grade Ajax Chemicals, Australia

Isopropyl-β-D-thiogalactoside (IPTG) Sigma Chemical Company, USA

Methanol Ajax Chemicals, Australia

Polyethylene glycol (average MW 20,000) BDH Laboratory Supplies, UK

Ponceau S Sigma Chemical Company, USA

Protein assay dye reagent Pierce Laboratories, USA prestained markers

Sodium azide (NaN₃) BDH Laboratory Supplies, UK

Sodium carbonate (Na₂CO₃) Ajax Chemicals, Australia

Sodium chloride (NaCl) Ajax Chemicals, Australia

Sodium dihydrogen orthophosphate Sigma Chemical Company, USA(NaH₂PO₄.2H₂O)

Sodium dodecyl sulphate (SDS) Bio-Rad Laboratories, USA

Sodium hydrogen carbonate (NaHCO₃) BDH Laboratory Supplies, UK

Sodium phosphate (Na₂HPO₄) Sigma Chemical Company, USA

N,N,N′,N′-Tetramethylethylene-diamine (TEMED) Bio-Rad Laboratories, USA

³H-thymidine (methyl) DuPont, USA

Trichloroacetic acid (TCA) BDH Laboratory Supplies, UK

Triethanolamine BDH Laboratory Supplies, UK

Tris (hydroxymethyl) aminomethane (Tris) BDH Laboratory Supplies, UK

Trypan blue Calbiochem, USA

Tryptone Becton Dickinson, USA

Tween 20 (Polyxyethylenesorbitan monolaurate) Sigma Chemical Company,USA

Yeast extract Sigma Chemical Company, USA

2. Buffers and Solutions

All buffers and solutions were prepared in Milli Q (Millipore, USA) H₂Ounless otherwise stated.

Acrylamide-Bis (50% w/v)

A commercially available premixed preweighed acrylamide/bis powder wasdiluted in 162 ml water (Bio-Rad Laboratories, USA) to yield a 50%solution. This was stored at room temperature, protected from light.

Ammonium Persulphate (10% w/v)

A 10% ammonium persulphate solution was prepared by adding 1 g ammoniumpersulphate to 10 ml H₂O. This solution was stored at 4° C. for up to3-4 days.

Binding Buffer

A 0.1 M Na₂HPO₄ solution, adjusted to pH 9.0, was prepared and stored atroom temperature.

10% Milk Powder

10 g of skim milk powder dissolved in 100 ml PBS.

BGG Standard for the Pierce Protein Assay

1.45 mg BGG was dissolved in 1 ml H₂0, stored at −20° C. in 100 μlaliquots and used for the standard curve in the Pierce protein assay(Pierce, USA) according to the manufacturer's instructions.

Coomassie Brilliant Blue R-250

A 0.2% (w/v) Coomassie Brilliant Blue R-250 (Coomassie Blue) solutionwas prepared by dissolving 0.2 g of Coomassie Brilliant Blue R-250 in100 ml of 50% (v/v) methanol, 10% (v/v) glacial acetic acid in H₂O. Thissolution was filtered through Whatman #1 filter paper and stored at roomtemperature in the dark.

Destaining Solution

Coomassie Blue stained gels were destained using 7% glacial acetic acid.The destaining solution was prepared by diluting 70 ml glacial aceticacid to 1 L with H₂O.

ELISA Coating Buffer, pH 9.6

This buffer consisted of 0.86 g Na₂CO₃ and 1.72 g NaHCO₃ dissolved in100 ml H₂O and was stored at 4° C. The pH of this solution was asindicated and did not need to be adjusted.

Elution Buffer

A 500 ml solution containing 500 mM imidazole, 50 mM NaH₂PO4, 300 mMNaCl, and 8 M urea was prepared and stored at room temperature.

FACS Wash Buffer (1% FBS/0.02% NaN3/PBS)

5 ml of FBS and 1.54 ml of 1 M NaN₃ solution was added to 443.46 ml ofPBS and the solution stored at 4° C.

FBS-15% DMSO

To 50 ml of FBS, 8.8 ml of DMSO was added to yield a 15% DMSO solution.This was aliquoted into 10 ml polypropylene tubes and stored at −20° C.until required.

Luria-Agar (L-Agar)

To 1 L of L-broth 15 g of agar was added and the solution sterilised byautoclaving.

Luria-Broth (L-Broth)

L-broth was prepared by the addition of 5 g of tryptone, 2.5 g of yeastextract and 2.5 g of NaCl to 450 ml of water. The pH was then adjustedto 7.0 and the solution made up to 500 ml. The medium was thensterilised by autoclaving.

Lysis Buffer

A 500 ml solution containing 100 mM NaH₂PO4, 10 mM Tris-HCl, 8 M urea,adjusted to a pH of 8.0 using HCl was prepared and stored at roomtemperature.

Phosphate Buffered Saline (PBS), pH 7.2

A 10× stock solution was prepared by dissolving 85 g NaCl, 3.9 gNaH₂PO₄.2H₂O and 10.7 g Na₂HPO₄ in 1 L H₂O. This was stored at roomtemperature and diluted ten-fold when required.

PBS/0.1% BSA

0.1 g of BSA was dissolved in 100 ml of PBS, and used on the day ofpreparation.

PBS/Tween 20 (0.05% v/v)

PBS/Tween 20 (0.05% v/v) was prepared by adding 2.5 ml Tween 20 to 5 LPBS. This solution was stored at room temperature.

Phytohaemagglutinin (PHA) (400 μg/ml)

2 mg of lyophilised PHA was reconstituted with 5 ml H₂O under sterileconditions to give a final concentration of 400 μg/ml. This was thenaliquoted into 50 μl lots and stored at −20° C.

Resolving Gel Buffer, pH 8.8

This buffer was prepared by adding 182 g Tris, 4.0 g SDS and H₂O to 950ml. The pH was adjusted to 8.8 with HCl and made up to 100 ml with H₂O.This buffer was stored at 4° C.

RPMI 1640/Penicillin-Streptamycin-Glutamine (Wash Medium)

5 ml of Penicllin-Streptamycin-Glutamine (Gibco, USA) was added to 500ml of RPMI 1640 medium to yield final concentrations of 2 mM L-glutamineand 100 U/ml of penicillin/streptamycin. The medium was stored at 4° C.protected from light.

RPMI 1640/10 U/ml Sodium Heparin (Heparinised Medium)

5000 units of sterile preservative free sodium heparin and 5 ml ofPenicillin-Streptamycin-Glutamine (Gibco, USA) was added to 500 ml ofRPMI 1640 medium to yield final concentrations of 10 U/ml sodiumheparin, 2 mM L-glutamine and 100 U/ml of penicillin/streptamycin. Themedium was stored at 4° C. protected from light.

RPMI 1640/5% Human AB+ Serum (Complete Medium)

To 500 ml of RPMI 1640 medium, heat inactivated human AB+ serum andPenicillin-Streptamycin-Glutamine (Gibco, USA) were added to yield finalconcentrations of 5% human serum, 2 mM L-glutamine and 100 U/ml ofpenicillin and streptamycin. The medium was stored at 4° C. protectedfrom light.

SDS Running Buffer

This buffer was prepared by dissolving 6.06 g Tris, 28.8 g glycine and 2g SDS in 2 L H₂O. This solution was stored at room temperature.

SDS Reducing Sample Buffer

A stock solution was prepared by the addition of 3.8 g Tris, 11.5 g SDS,50 ml glycerol and 100 mM (1.56 g) DDT to 500 ml H₂O. 0.1% bromophenolblue was added to allow tracking of the protein dye front. The bufferwas stored at −20° C. and thawed just prior to use .

1 M Sodium Azide

6.5 g of NaN₃ was carefully added to 100 ml of H₂O in a fume hood andthe resulting solution stored at room temperature.

Stacking Gel Buffer, pH 6.8

This buffer was prepared by the addition of 30 g Tris and 2.0 g SDS to450 ml H₂O. Following adjustment of the pH with HCl the volume was madeup to 500 ml with H₂O and the buffer stored at 4° C.

Transfer Buffer

This buffer was prepared by adding 5.81 g Tris, 2.93 g glycine, 0.375 gSDS to 800 ml H₂O. This solution was stored at room temperature in thedark.

Trypan Blue (0.25% w/v)

This stain was prepared by dissolving 0.25 g trypan blue in 100 ml H₂O.This was filtered through Whatman #I filter paper and stored at roomtemperature.

Wash Buffer

A 500 ml solution containing 50 mM imidazole, 50 mM NaH₂PO4, 300 mMNaCl, and 8 M urea was prepared and stored at room temperature.

3. Study Population

(a) Study Population

Peanut and tree nut allergic individuals were recruited from the AlfredHospital Allergy Clinic and the Department of Pathology and Immunology,Monash University. Donors were chosen on the basis of a history ofclinical symptoms of nut allergy, positive peanut or tree nut specificIgE (CAP-Pharmacia, score>1) or skin prick test (wheal>5 mm). The studywas approved by the Alfred Hospital Ethics Committee and informedconsent was obtained from all donors before blood was obtained.

(b) Protein Concentration Determination

To determine the protein concentration of nut and seed extracts a Piercemicro protein assay, based upon bicinchoninic acid induced detection ofprotein induced reduction of cuprous ion, was used (Smith et al. 1985).A 1450 μg/ml stock of BGG was used to establish a standard curve in therange of 725-6 μg/ml. Samples for testing and BGG standard were dilutedin MilliQ water and plated in triplicate (25 μl/well) in a 96 well flatbottom plate (Linbro, USA). 200 μl of Pierce Micro-Protein Assay dyeconcentrate was added to each well and mixed well. Plates were then readin a Bio-Rad 3550 microplate reader (Bio-Rad Laboratories, USA) at 595nm and sample concentrations extrapolated from the standard curve usingMicroplate manager software.

(c) Endotoxin Estimation

Endotoxin estimation was performed using a Biowhittaker PyrogentMulti-test kit (Cambrex, USA) as per the manufacturer's instructions.

(d) Antigens

Preparation of Crude Peanut Extract, Ara h 1 and Ara h 2

Preparation of crude peanut extract, purification of natural Ara h 1 andAra h 2 fractions, and expression of recombinant Ara h 2 is described inexample 1.

Ara h 2Peptides

All Ara h 2 peptides were synthesized based on the published amino acidsequence deduced from the cDNA sequence of Ara h 2 (Stanley et al.1997). The Ara h 2 20-mer peptides (11-18 amino acid overlap) werepurchased from Mimotopes, Clayton, Australia. Lyophilised peptides werereconstituted in sterile PBS to a concentration of 1 mg/ml and filtersterilised by passage through a 0.2 μm sterile filter. Hydrophobicpeptides which would not reconstitute properly were first dissolved in asmall volume (10 μl/mg peptide) of DMSO and then brought to theappropriate volume with sterile PBS and sterilised as above.

(e) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Gel Staining

Preparation of 14% SDS-polyacrylamide Mini Gels

A 14% resolving gel solution was prepared by mixing 7 ml 50%acrylamide/BIS, 9.4 ml resolving bel buffer, 250 μl 10% SDS, 7.7 mlwater, 6.25 μl TEMED and 625 μl ammonium persulphate. The gel solutionwas transferred into a minigel casting unit (Novex, USA) up to a pointapproximately 2 cm from the top of the gel plates. H₂O was then overlaidonto the gel solution to produce a flat gel surface and excludeatmospheric oxygen which interferes with polymerisation. Upon gelpolymerisation (approximately 60 minutes), the gel surface was rinsedwith H₂O and a 4% stacking gel added. A solution sufficient for 4minigels comprised 4.2 ml stacking gel buffer, 1.0 ml 50% acrylamide,4.2 ml stacking gel buffer, 125 μl of 10% SDS, 6.3 ml H₂0, 5.0 μl TEMEDand 1 ml ammonium persulphate. A plastic template was then inserted intothe stacking gel and the gel was allowed to stand for 30 minutes topolymerise. If not used immediately, gels were stored at 4° C. in amoistened sealed plastic bag for up to 14 days.

SDS-PAGE

Gels were loaded on an electrophoresis unit (Novex, USA) and runningbuffer was added to the inner and outer chambers of the tank. Sampleswere prepared by diluting to a maximum concentration of 1.5 mg/ml inSDS-sample buffer and water and boiled for 5 minutes to ensure proteinreduction, before loading onto gels. Generally 20 μl (for 10 well gels)and 10 μl (for 15 well gels) of sample was loaded per well. WhereSDS-PAGE was being carried out for nitrocellulose transfer of Ara h 1,the Ara h 1 solution was diluted I in 10, to avoid non-specific bindingof serum IgE. Low molecular weight standards (Invitrogen, USA) for gelsto be stained with Coomassie Brilliant Blue were inserted at a volume of5 Ill/well and 8 μl/ml for nitrocellulose transfer. Gels wereelectrophoresed at a constant current of 125 volts per gel until the dyefront reached the bottom of the gel.

Coomassie Brilliant Blue Staining of SDS-PAGE Gels

Gels were stained in 0.2% Coomassie Brilliant Blue for 1 hour at roomtemperature and destained in destaining solution at room temperature.

(f) Immunoblotting

SDS-PAGE gels were assembled into stacks in transfer buffer as follows:blotting paper, nitrocellulose membrane, gel and blotting paper. Sampleproteins and pre-stained molecular weight markers wereelectrophoretically transferred from SDS-PAGE gels to nitrocellulose at30 volts for 3 hours in an Xcell II blot module (Novex, USA). Followingtransfer, the nitrocellulose membrane was stained with Ponceau S toensure adequate protein transfer, then cut into strips and blocked byincubation with 10% milk powder-PBS for 1 hour at room temperature. Theblocked nitrocellulose membrane was then washed in PBS and incubatedwith sample sera diluted 1 in 5 with 1% milk powder-PBS-Tween for allproteins except for Ara h 1, whereupon a 1 in 10 serum dilution wasused. Incubation took place overnight, following which the blot waswashed 3 times in PBS-Tween, 5 minutes/wash. A rabbit anti-human IgEHRPO conjugate (Dako, Denmark) diluted 1 in 500 dilution in 1% milkpowder-PBS-Tween was then applied and incubated 1-2 hours at roomtemperature. Following washing 3 times in PBS-Tween, then 3 times in PBSfor 5 minutes/wash, the membrane was incubated for 1 minute with freshlyprepared chemiluminescence substrate (Du Pont, USA). Excess reagent wasdrained and the blot placed between two sheets of plastic transparencyfilm. The nitrocellulose was then photographed and analysed usingLabworks image acquisition software (UVP Laboratory Products, UK).

(g) Inhibition Immunoblotting

To explore IgE cross-reactivity, inhibition immunoblotting was carriedout. Sera for immunoblotting were diluted 1 in 5 with 1% milk powderPBS-0.05% Tween and incubated with a range of concentrations of antigen(25-100 μg/ml sera) for two hours. To exclude false reduction in IgEbinding in subsequent immunoblotting experiments via protease induceddestruction, protease inhibitor tablets were added to the diluent(Roche, Germany). Immunoblotting was then carried out as describedabove.

(h) Cell Culture

Cryopreservation and Thawing of PBMCs and TCLs

PBMC and TCL were cryopreserved in FBS/15% DMSO in polypropylenecryovials (Greiner, Germany). Following pelleting by centrifugation,329×g, 10 minutes, cells were resuspended in ice cold FBS/15% DMSO at aconcentration of 0.5-1×10⁷ cells/ml. Vials were placed into a “MrFrosty” freezing container (Nalgene, USA), and placed in a −80° C.freezer overnight before transfer to liquid nitrogen (−180° C.) for longterm storage. Thawing took place via placement of the frozen cellsuspension within a 37° C. water bath. When thawed, the suspension wastransferred to a 25 ml tube and wash medium was added drop-wise withcontinual mixing until 15 ml had been added. Cells were recovered bycentrifugation at 329×g for 10 minutes followed by resuspension incomplete medium.

Mononuclear Cell Separation from Peripheral Blood

Peripheral blood (80-100 ml) was collected by venipuncture in 50 mlsyringes flushed with preservative free sodium heparin to preventclotting. Blood was then diluted 1:1 with warm (37° C.) heparinisedmedium. 25 ml of diluted blood was gently layered onto 15 ml ofFicoll-Paque in a 50 ml polypropylene tube. Samples were centrifuged at725×g for 25 minutes at room temperature, brake off. The PBMC layer(buffy coat) was harvested using a sterile disposable plastic pipette,placed into a fresh 50 ml tube and washed with RPMI/heparin medium (30ml RPMI:20 ml PBMC). Cells were pelleted at 500×g for 15 minutes andwashed in 20 ml of plain RPMI-1640, 329×g for 10 minutes. PBMC were thenresuspended in RPMI/5% human AB⁺ serum (complete medium) and the viablecell number determined by trypan blue exclusion using a haemocytometer.These cells were then used for culture immediately of cryopreserved asdescribed above.

PBMC Proliferation Assays

1×10⁶ PBMC/well were cultured in 96-well U bottom plates (Linbro ICNBiomedicals, USA) in 200 μl complete medium along with antigen, mediumalone, tetanus toxoid and PHA (2 μg/ml) as negative and two positivecontrols respectively for 7 days at 37° C. in a humidified incubator, 5%CO₂. Cultures were pulsed for the last 16 hours with ³H-thymidine (1μCi/well) and harvested onto printed glass fibre filters (Wallac, U.K.)using a 96 well automatic cell harvester (Skatron, UK). ³H-thymidineincorporation was measured by liquid scintillation spectroscopy with aWallac 1205 β-counter.

Generation of Short Term T Cell Lines

Freshly harvested or frozen stored PBMC were cultured in 24-well plates(Greiner Biotechnik, Germany) at 2.5×10⁶ cells per well (2 ml volume) incomplete medium with antigen at an optimised concentration for 7 days at37° C. in a 5% CO₂ humidified incubator. In the case of frozen PBMC thecells were first washed once in wash medium, 1 in 10 dilution of PBMC tomedium, and pelleted at 329×g for 10 minutes to remove DMSO. At day 7cells were washed once and resuspended at 1 to 1.5×10⁶/ml and addedtogether with 1×10⁶/ml washed irradiated (3000 rads; Gammacell 1000Elite, Nordion International, Inc.) autologous PBMC (from liquidnitrogen stocks) and antigen into fresh 24-well plates. At day 2following restimulation, 25 U/ml of recombinant human interleukin-2(rIL-2) was added and at day 4, 1 ml of culture medium was removed andreplaced with fresh medium and 25 U/ml rIL-2. For some experiments dueto low cell numbers at 2 weeks, 3 week TCLs were generated byrestimulation with antigen and rIL-2 as above for a further week. In allexperiments T cells were rested for 6 to 7 days after the last additionof antigen and APC.

Short Term T Cell Line Proliferation Assays

5.0×10⁴/well TCL cells were cultured in triplicate in 96-well U bottomplates (Linbro ICN Biomedicals, USA) with antigen or medium alone andrIL-2 as negative and positive controls respectively, in the presence ofautologous irradiated (3000 rads) PBMC (5.0×10⁴/well). Cultures wereincubated for 3 days. In the last 16 hours of culture, wells were pulsedwith ³H-thymidine (1 μCi/well), then harvested onto printed glass fibrefilters with a 96-well automatic cell harvester. ³H-thymidineincorporation was measured by liquid scintillation spectroscopy.

Assessment of Tissue Culture Reagent Mitogenicity and Toxicity

Mitogenic and toxic potential of tissue culture antigens as well ashuman sera used for preparation of complete medium was assessed usingshort term house dust mite specific TCL generated in a manner analogousto nut specific TCL. Mitogenicity was assessed via 3 days stimulation ofthis TCL with increasing concentrations of the relevant reagent, in thepresence of autologous irradiated PBMC as APC, along with control wellscontaining either cells alone or house dust mite and IL-2 as negativeand two positive controls respectively. For the last 16 hours ofculture, cells were pulsed with ³H-thymidine (1 μCi/well) and harvestedonto printed glass fibre filters with a 96-well automatic cellharvester. In the last 16 hours of culture, wells were pulsed with³H-thymidine (1 μCi/well), then harvested onto printed glass fibrefilters with a 96-well automatic cell harvester. ³H-thymidineincorporation was measured by liquid scintillation spectroscopy.Toxicity was assessed in a similar manner, except that, in addition toantigen, cells were co-cultured with 25 U/ml of rIL-2, in the absence ofAPC.

Harvesting of T Cell Culture Supernatants for Cytokine Testing

Supernatants (70-75 μl/well of triplicate cultures) were harvested andpooled from T cell proliferation assay cultures at 48 hours (IL-5,IFN-γ) and frozen at −80° C. Harvested supernatants were replaced withwarm (37° C.) complete medium and cells subsequently pulsed andharvested as described previously.

(i) Flow Cytometry

T cells from in vitro culture were washed once in cold (4° C.) FACS washbuffer prior to staining. Cells (0.5×10⁶/tube) were stained withappropriate fluorochrome-labelled monoclonal antibodies or relevantisotype controls (10 μl/tube) for 15 minutes on ice and protected fromlight. Cells were washed once by addition of cold wash buffer, pelletedby centrifugation (329×g 5 minutes, 4° C.) and resuspended in washbuffer. The percentage and mean fluorescence intensity of stained cellswas determined from 100,000 events using a Becton Dickinson FACScaliburflow cytometer and “Cell Quest” software.

(j) Cytokine ELISA

IL-5 and IFN-γ levels in culture supernatants were measured by sandwichELISA. White Costar (Corning, USA) ELISA plates were coated with capturemAb (IL-5 and IFN-γ, 2 μg/ml; 30μl/well) diluted in binding bufferovernight at 4° C. Plates were then washed three times in PBS/0.05%Tween (wash buffer) and wells blocked with 100 μl/well of 1% BSA/PBS(blocking buffer) for 1 hour at room temperature. Following three washesin wash buffer, 30 μl/well of serial dilutions of recombinant human IL-5or IFN-γ (5000-0.15 pg/ml) in blocking buffer-0.05% Tween or culturesupernatants were added and incubated overnight at 4° C. Following fourwashes in wash buffer, plates were incubated with biotinylated detectionmAb (IL-5, 1 μg/ml; IFN-γ, 0.5 μg/ml; 50 μl/well) diluted in blockingbuffer for 1 hour at room temperature. Plates were then washed 6 timesin wash buffer and incubated with a 1 in 2000 dilution ofstreptavidin-peroxidase (50 μl/well) in blocking buffer for 45 minutesat room temperature. Following 8 washes in wash buffer, 100 μl/well offreshly prepared chemiluminescent substrate (Perkin-Elmer, USA) was usedand plates read in a Lumicount microplate glow luminometer (PackardInstrument Company, USA), 0.5 seconds/well, automatic sensitivitysetting. Standard curve construction and determination of unknowncytokine levels was performed using Packard Instruments software. IL-5and IFN-γ ELISA sensitivities were 2 pg/ml and 4 pg/ml, respectively.

(k) Statistical Analysis

All statistical analysis was performed using SPSS statistical software(SPSS, USA). For non-normally distributed data, a Mann-Whitney test wasemployed to assess the level of significance of differences betweenvalues for a particular parameter for any two groups. For continuousvariables, linear regression analysis (Pearson's) was performed toassess the degree of correlation between two parameters while logisticregression analysis was performed for categorical data.

EXAMPLE 2

Subject Characterisation

Clinical characteristics of donor subjects used for these experimentsare detailed in table 4. A total of 22 peanut allergic subjects wereused for Ara h 2 T cell epitope mapping, of whom 15 were female. Theaverage age of subjects was 32 years (range 19-55 years). Nineteen ofthe 22 subjects suffered from other allergic diseases, including asthma,eczema or allergic rhinitis. Of the 19 subjects for whom skin pricktesting had been performed, only one was non-atopic. Each subjectdescribed typical features of anaphylaxis on exposure to peanut,beginning within minutes of that exposure. Laryngeal oedema was the mostcommon symptom, with 17 of the 22 subjects describing this symptom atthe time of anaphylaxis. Other common symptoms were asthma, generalisedurticaria, and facial angioedema. Five of the 22 subjects reportedanaphylaxis occurring only on exposure to peanut, but only one of thosefive was also RAST test negative to all other nuts tested. RAST testingdata showed no correlation to the severity of reactions subjectsexperienced with exposure to peanut.

Peanut Allergen Preparation

The manufacture and characterisation of CPE and recombinant Ara h 2 isdescribed in Example 1. The sequences of the Ara h 2 peptides used forepitope mapping are illustrated in FIG. 1 (Chiron Mimitopes, Australia).Each peptide was 20 amino acids in length with an 11 amino acid sharedsequence with adjacent peptides, except for that closest to theN-terminus, where the matching sequence was 18 amino acids in length.Characterisation of peptides with regards T cell mitogenicity andtoxicity is displayed in FIG. 2, and demonstrates that all peptides werefree of these potential confounders.

Western Blotting of Ara h 2

To determine the frequency of Ara h 2 IgE reactivity amongst the studycohort, western blotting of CPE was carried out. These results are shownin FIG. 3 and demonstrate that 20 out of the 22 peanut allergic subjectspossessed IgE reactive to a doublet of approximately 14 kDa,representing Ara h 2. Interestingly, apart from Ara h 2, 19 of the 22subjects recognised a band of approximately 11 kDa, likely to representeither Ara h 3, Ara h 5 or Ara h 6. Non-peanut allergic controls alsoshowed binding to Ara h 2 and the 11 kDa protein, but this was weak andpotentially non-specific in this highly sensitive assay.

Polyclonal T Cell Responses to Crude Peanut Extract

To ensure that CPE-specific T cells were present within the studysubjects' T cell repertoire, PBMC proliferative responses to stimulationwith CPE were analysed, demonstrating a dose response for both peanutallergic subjects and non-peanut allergic controls. For each subject,the lowest dose that produced maximal stimulation at 7 days was used todrive peanut specific T cell lines for use in peptide assays, and rangedbetween 50 and 200 μg/ml.

Mapping of Ara h 2 T Cell Epitopes

To determine the T cell epitopes of Ara h 2, oligoclonal CPE-specificTCL were generated from PBMC of 21 peanut-allergic donors and 5non-peanut allergic donors, and stimulated with a nested set ofsynthetic peptides spanning the entire Ara h 2 sequence (FIG. 1). Apeptide concentration of 10 μg/ml was determined to be the optimalstimulating concentration within proliferation assays. Optimisationincluded assays at both 10 μg/ml and 30 μg/ml. Responses to eachconcentration proved similar, although some subjects who did not respondsignificantly to any peptide at a concentration of 10 μg/ml demonstratedan indiscriminate low grade response to most peptides at the higherconcentrations, that did not provide any differential signal. In thissetting, and because of the limited availability of donor cells and thecost of commercially prepared peptides, the lower concentration wasused.

Several approaches to the production of TCL were used, includingstimulation of cells with two and three pulses of CPE separated by 7days each, two pulses of CPE with a third pulse of rAra h 2, againseparated by 7 days each, and three stimulations with rAra h 2.Responses by TCL receiving two CPE stimulations were in general lowerthan those receiving three stimulations, such that it was felt thesensitivity of the assay may be compromised. Responses of TCL receivinga third pulse with rAra h 2 were surprisingly infrequent on the basis ofthe frequent sensitivity demonstrated to Ara h 2 by western blotting,while responses by cells receiving three pulses with rAra h 2 wereuniversally absent, suggesting that the rAra h 2 extract may havecontained other antigens or substances blunting the T cell response tothe allergen. This lead to the use of TCL stimulated with 3 pulses ofCPE, each separated by 7 days for all subjects tested. Data fromsubjects demonstrating a peptide response to two pulses of CPE and afinal pulse of rAra h 2 are also presented.

Individual responses to peptide, Ara h 2 and CPE are demonstrated inFIGS. 4 and 5 and summarised in table 5. A total of 9 (41%) of the 22peanut allergic subjects demonstrated a proliferative response to one ormore of the Ara h 2 peptides, with 8 subjects responding using CPEdriven TCL, two subjects responding using both methods of TCLgeneration, and one subject responding only when using a TCL driven witha pulse of rAra h 2. Of interest, PBMC from non-peanut allergic subjectscould not be used to generate a TCL, cells becoming non-viable after twostimulations or demonstrating a non-discriminatory “high background”response to all antigens assayed. CPE specific TCL could be generatedfrom 19 (86%) of the 22 peanut allergic subjects, with 6 (32%) of those19 peanut specific TCL responding to rAra h 2. Proliferative responsesto rAra h 2 showed best correlation with responses to Ara h 2 (19-38)and Ara h 2 (28-47), in that where a subject had a response to thesepeptides, 60% also had a proliferative response to Ara h 2. No otherpeptides were as clearly associated with a response to rAra h 2. Only 2peptide non-responsive TCL demonstrated a proliferative response to rArah 2. No subject without specific IgE towards Ara h 2 demonstrated aproliferative response to peptide, but the intensity of IgE reactivitycould not otherwise be used to predict a peptide response.

Using CPE driven TCL, of the 17 peptides tested, 7 (41 %) induced aproliferative response. Ara h 2 (19-38) and Ara h 2 (73-92) wereassociated with the greatest frequency of response, producingproliferative responses in 3 of the 8 responders. Other peptidesinducing proliferative responses were located at Ara h 2 (28-47), Ara h2 (55-74), Ara h 2 (82-101), Ara h 2 (91-110), Ara h 2 (100-119), andAra h 2 (136-155). No peptide was associated with a response in over 25%of the 22 CPE driven TCL. Only 2 peptides produced responses in greaterthan 25% of CPE driven TCL that demonstrated a peptide response, thesebeing Ara h 2 (19-38) and Ara h 2 (73-92). Comparison of the magnitudeof response to each antigen demonstrates a great variation betweensubjects. When response magnitude was ranked for each subject, thegreatest responses were towards Ara h 2 (19-38), with 3 subjectsdemonstrating their greatest response to this peptide.

Peptide responses by rAra h 2 pulsed TCL occurred in 3 of 20 TCLgenerated in this fashion. Peptide responses were widespread amongstthose responders, and only two peptides, Ara h 2 (64-83) and Ara h 2(109-128) did not produce a response in at least one subject. Themagnitude of responses towards peptides by these TCL was greater thanthat demonstrated by CPE driven TCL, the greatest response being bysubject 10 towards Ara h 2 (37-56), where the stimulation index was30.3. Comparison of peptide responses demonstrated by the differenttypes of TCL revealed some common peptide responses, but severaldifferences. Only two subjects demonstrated significant peptideresponses to both methods of TCL preparation. Subject 10 reacted to Arah 2 (19-38) using both types of TCL preparation, but the magnitude ofthe response to this peptide in the rAra h 2 pulsed TCL was less thanthat generated by stimulation with Ara h 2 (37-56) and Ara h 2 (46-65).The pattern of reactivity demonstrated by subject 6 was similar for bothtypes of TCL, but did not include Ara h 2 (73-92) or Ara h 2 (136-155)for rAra h 2 pulsed TCL.

Cytokine Responses to Peptides Associated with a Proliferative Response

To identify the phenotype of T cells associated with a peptideproliferative response, supernatants were collected from TCL cultures 48hours after stimulation with peptides and assayed for the presence ofIL-5 and IFN-γ. For each donor, peptides associated with a proliferativeresponse and two peptides not associated with a proliferative responsewere tested. Of the 8 CPE driven TCL examined, 7 demonstrated detectablecytokine levels. Individual cytokine ratios for CPE driven TCL aredemonstrated in FIG. 5.8. The magnitude of cytokine responses associatedwith individual peptides varied greatly between subjects, from the lowerlimits of detection for both cytokines, up to 2054 pg/ml for IL-5, and2966 pg/ml for IFN-γ. In general, cytokine responses were at the lowerlimits of detection. The greatest individual IL-5 response was to Ara h2 (19-38) with subject 21 producing 2054 pg/ml of IL-5 and subject 10producing 540 pg/ml of IL-5 towards this peptide. These responses weresubstantially greater than the next largest cytokine responsedemonstrated by other subjects.

IL-5/IFNγ ratios were skewed towards greater IL-5 production, forpeptide responses in 5 of the 7 subjects whose CPE driven TCLdemonstrated a proliferative response. The greatest ratio occurredtowards Ara h 2 (19-38), being approximately 20 for subject 21, and 7 insubject 10. Only these subjects demonstrated an IL-5/IFN-γ ratio ofgreater than 1 for rAra h 2 and CPE. For peptides not associated with aproliferative response, the IL-5/IFN-γ ratio was less than 1 for 5 outof 7 subjects.

Cytokine responses by rAra h 2 pulsed TCL could only be demonstrated intwo of the three demonstrating a proliferative response to peptides andare illustrated in FIG. 5.8. The response of these TCL was predominantlycharacterised by IFN-γ production (data not shown), again suggestingthat rAra h 2 pulsed TCL were of a different phenotype to CPE generatedTCL, and may have been contaminated with either different antigens orother immunomodulatory substances. IL-5 production could only bedetected in low levels or not at all for peptides not associated with aproliferative response, but was clearly detectable for those producingproliferation. This is in contrast to IFN-γ, for which detectable levelsoccurred for all peptides, although at increased levels for thosepeptides producing a proliferative response.

EXAMPLE 3 Site-Directed Mutagenesis of ARA H 2

Site directed mutagenesis of a Ara h 2 construct is carried out usingthe QuickChange™XL site direct mutagenesis kit (Stratagene, Calif., USA)according to the manufacturer's instructions. Primers are used toreplace one or more cysteine amino acids with an alanine. Aftersite-directed mutagenesis the construct is sequenced to confirm thenucleotide changes. The construct containing the mutant Ara h 2 istransformed into E. coli strain BL21-CodonPlus® (DE3)-RIL competentcells, expressed and purified as above. This technique can becorrespondingly applied to the mutation of any other type of amino acid.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.TABLE 4 Clinical features of the 22 peanut allergic subjects used for Tcell Ara h 2 epitope mapping Age at first Time since Allergic Known nutOther food reaction last reaction Subject Sex Age disease Clinicalfeatures allergens allergens (yrs) (mths) level score Atopic status  1 F27 asthma, laryngeal oedema, urticaria, peanut, hazelnut, sesame 10 22.07 2 GP, B, HDM, rhinitis, facial angioedema walnut seed, baked C, Aeczema beans  2 F 34 asthma asthma, laryngeal oedema, peanut peas,lentils 14 192 0.73 2 GP, B, HDM urticaria, facial angioedema  3 F 40asthma, asthma, laryngeal oedema, peanut, Brazil nut, nil 2 12 3.61 3 Ceczema loss of consciousness, cashew nut, urticaria, facial angioedemahazelnut  4 M 27 rhinitis asthma, laryngeal oedema, peanut, almond, pinenuts, 2 4 mixed nut urticaria brazilnut, hazelnut, citrus seeds RAST: ¾macadamia, walnut  5 F 31 rhinitis, GIT upset, asthma, laryngeal peanut,almond, 1.5 18 13.6 3 GP, HDM, C eczema oedema, urticaria hazelnut,pistachio  6 F 33 nil GIT upset, urticaria peanut, hazelnut pine nuts0.5 3 13.3 3 GP, B, HDM, C  7 F 27 eczema asthma, laryngeal oedema,peanut, almond 19 11 9.53 3 urticaria, angioedema  8 F 19 asthmalaryngeal oedema, urticaria peanut, almond, crustacea 3 12 3.09 2 GP,HDM, C Brazil nut, cashew, hazelnut, walnut  9 M 29 asthma, GIT upset,laryngeal oedema, peanut, almond, pine nuts 1.5 36 1.22 2 GP, B, HDM,rhinitis, facial angioedema Brazil nut, cashew, C eczema hazelnut,walnut Age at Peanut RAST first Time since Allergic Known nut Other foodreaction last reaction Subject Sex Age disease Clinical featuresallergens allergens (yrs) (mths) level score Atopic status 10 F 49 nilGIT upset, asthma, laryngeal peanut, cashew nut peas 2 18 17.6 4 GPoedema, loss of consciousness, urticaria 11 M 30 asthma GIT upset,asthma, laryngeal peanut, hazelnut 12 18 0.51 1 oedema, facialangioedema 12 F 22 asthma laryngeal oedema, urticaria, peanut 0.5 2416.3 3 GP, HDM facial angioedema 13 F 36 asthma, GIT upset, asthma,peanut, hazelnut 4 2 3.09 2 GP, HDM, C eczema hypotension, facialangioedema 14 F 50 nil laryngeal oedema, urticaria, peanut 18.5 21 6.873 GP, HDM, C facial angioedema 15 M 27 asthma, asthma, laryngeal oedema,peanut, almond, avocado 1.5 24 12.4 3 GP, B, HDM rhinitis urticaria,facial angioedema hazelnut 16 F 37 rhinitis, GIT upset, asthma,laryngeal peanut, hazelnut pine nuts 10 36 0 0 GP, HDM, eczema oedema,hypotension peanut 14 mm 17 M 35 asthma, asthma, urticaria, facialpeanut, hazelnut 8 12 2.82 2 rhinitis, angioedema eczema 18 F 22rhinitis GIT upset, asthma, laryngeal peanut, hazelnut, 8 9 0.39 1 GPoedema, facial angioedema pistachio 19 M 55 asthma, asthma, laryngealoedema, peanut, walnut banana 1.5 120 2.01 2 GP, HDM, A eczema facialangioedema 20 F 30 asthma asthma, uticaria, laryngeal peanut, Brazilnut, 3 228 0 oedema, facial angioedema almond 21 M 28 asthma GIT upset,laryngeal oedema, peanut, hazelnut, 5 1 100 6 HDM urticaria egg, milk 22F 32 asthma, asthma, urticaria, facial peanut, 0 0 0 eczema angioedemaLegend:GP, grass pollen;B, birch;HDM, house dust mite;C, cat;A, alternaria

TABLE 5 Summary of TCL proliferative responses to Ara h 2 peptides, rArah 2, and CPE

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1. An isolated peptide comprising an Ara h 2 T cell epitope said peptidecomprising at least 5 contiguous amino acids of an amino acid sequencederived or selected from: (i) amino acids 19-74 (ii). amino acids73-137; (iii) amino acids 127-155; inclusive, of Ara h 2 or homologuethereof; and wherein said peptide molecule is capable of interactingwith T cells and modifying T cell function when incubated with cellsfrom subjects having a condition characterised by an aberrant, unwantedor otherwise inappropriate immune response to Ara h 2 or a derivative,homologue, mutant, chemical equivalent or mimetic of said peptide. 2.The isolated peptide according to claim 1 wherein said peptide is from5-20 amino acids in length.
 3. The isolated peptide according to claim 2wherein said amino acid sequence is derived or selected from amino acids19-74 inclusive of Ara h 2 or a homolog thereof.
 4. The isolated peptideaccording to claim 2 wherein said amino acid sequence is derived orselected from amino acids 28-47 inclusive of Ara h 2 or a homologthereof.
 5. The isolated peptide according to claim 2 wherein said aminoacid sequence is derived or selected from amino acids 37-56 inclusive ofAra h 2 or a homolog thereof.
 6. The isolated peptide according to claim2 wherein said amino acid sequence is derived or selected from aminoacids 46-65 inclusive of Ara h 2 or a homolog thereof.
 7. The isolatedpeptide according to claim 2 wherein said amino acid sequence is derivedor selected from amino acids 55-74 inclusive of Ara h 2 or a homologthereof.
 8. The isolated peptide according to claim 2 wherein said aminoacid sequence is derived or selected from amino acids 73-92 inclusive ofAra h 2 or a homolog thereof.
 9. The isolated peptide according to claim2 wherein said amino acid sequence is derived or selected from aminoacids 91 -110 inclusive of Ara h 2 or a homolog thereof.
 10. Theisolated peptide according to claim 2 wherein said amino acid sequenceis derived or selected from amino acids 100-119 inclusive of Ara h 2 ora homolog thereof.
 11. The isolated peptide according to claim 2 whereinsaid amino acid sequence is derived or selected from amino acids 118-137inclusive of Ara h 2 or a homolog thereof.
 12. The isolated peptideaccording to claim 2 wherein said amino acid sequence is derived orselected from amino acids 127-146 inclusive of Ara h 2 or a homologthereof.
 13. The isolated peptide according to claim 2 wherein saidamino acid sequence is derived or selected from amino acids 136-155inclusive of Ara h 2 or a homolog thereof.
 14. The isolated peptideaccording to claim 2 wherein said amino acid sequence comprises at least5 amino acids derived from amino acid sequences selected from:RQQWELQGDRRCQSQLERAN (SEQ ID NO:4) RRCQSQLERANLRPCEQHLM (SEQ ID NO:5)PYDRRGAGSSQHQERCCNEL; (SEQ ID NO:10) and ELNEFENNQRCMCEALQQIM. (SEQ IDNO:12)


15. The peptide according to claim 14 wherein said amino acid sequenceis derived from SEQ ID NO:4.
 16. The peptide according to claim 14wherein said amino acid sequence is derived from SEQ ID NO:5.
 17. Thepeptide according to any one of claims 1-16 wherein said modification ofT cell functioning is the induction of T cell differentiation.
 18. Thepeptide according to claim 1 wherein said peptide exhibits reduced orablated IgE binding.
 19. An antibody directed to an isolated peptide ofany one of claims 1-17.
 20. The antibody according to claim 19 whereinsaid antibody is a polyclonal antibody.
 21. The antibody according toclaim 19 wherein said antibody is a monoclonal antibody.
 22. An isolatednucleic acid sequence encoding or complementary to a sequence encodingthe isolated peptide according to any one of claims 1-18.
 23. A methodfor the treatment and/or prophylaxis of a condition in a subject, whichcondition is characterised by an aberrant, unwanted or otherwiseinappropriate immune response to Ara h 2 or functional homologuethereof, said method comprising administering to said subject aneffective amount of a peptide according to any one of claims 1-18, anantibody according to any one of claims 19-21 or a nucleic acid moleculeof claim 22 for a time and under conditions sufficient to remove orreduce the presence or function in said subject of T cells directed tosaid Ara h
 2. 24. The method according to claim 23 wherein saidcondition is hypersensitivity to peanuts or tree nuts which contain Arah
 2. 25. The method according to claim 24 wherein said tree nuts arehazelnuts, almonds or Brazil nuts.
 26. Use of a peptide according to anyone of claims 1-18, an antibody of any one of claims 18-20 or a nucleicacid molecule of claim 21 in the manufacture of a medicament for thetreatment of a condition in a mammal which condition is characterised byan aberrant, unwanted or otherwise inappropriate immune response to Arah 2 or functional homologue thereof.
 27. Use according to claim 26wherein said condition is hypersensitivity to peanuts or tree nuts whichcontain Ara h
 2. 28. Use according to claim 27 wherein said tree nutsare hazelnuts, almonds or Brazil nuts.
 29. A pharmaceutical compositioncomprising one or more peptides according to any one of claims 1-18,antibodies of any one of claims 19-21 or nucleic acid molecules of claim21 together with one or more pharmaceutically acceptable carriers and/ordiluents.
 30. A method of diagnosing or monitoring a condition in amammal, which condition is characterised by an aberrant, unwanted orinappropriate response to Ara h 2, said method comprising screening forAra h 2 reactive T cells and/or antibodies utilising the peptidesaccording to any one of claims 1-18.
 31. The method according to claim30 wherein said condition is hypersensitivity to peanuts or tree nutswhich contain Ara h
 2. 32. The method according to claim 31 wherein saidtree nuts are hazelnuts, almonds or Brazil nuts.
 33. A method ofqualitatively and/or quantitatively detecting Ara h 2, or peptidesthereof, in a sample said method comprising screening for said Ara h 2or peptides thereof utilising an antibody according to any one of claims19-21.
 34. A diagnostic kit for use in the method of any one of claims29-32 wherein said kit comprises a peptide, antibody or nucleic acidmolecule according to any one of claims 1-22.